1. Field of the Invention
This invention relates to the production of peptides in the milk of transgenic mammals, particularly non-human placental mammals.
2. Related Art
Polymers of naturally occurring amino acids concatenated via their amino and carboxyl groups form the basis of many different biologically important compounds. Polymers of 3 to 100 amino acids are generally called peptides whilst larger concatamers are termed proteins. This is a purely arbitrary distinction, and the term "peptide" will be generally used throughout this specification even though the definition of peptides is not restricted to polymers of any particular size. Peptides can be biologically active without further modification or they can form the building blocks for more complex molecules by chemical incorporation into larger structures or by modification such as glycosylation. The term "peptide" is used herein to include biologically active or inactive molecules which may or may not be further modified by either chemical methods or in biological systems.
The direct chemical synthesis of peptides is expensive due to the cost of reagents and the high degree of purification needed to remove failed sequences. Microbial synthesis by recombinant DNA technology is not always appropriate for peptides, because of difficulties in their extraction and purification and the absence in the microbial host of enzymes for performing appropriate and correct post-translational modification. Heterologous proteins can be produced in stably transfected mammalian cell lines. Many such cell lines are available today and are used commercially, but concern remains that the cell lines were in general established from tumours of various types. More recently, the production of proteins in the milk of transgenic mammals such as sheep has become a reality, as illustrated in WO-A-8800239 and WO-A-9005188.
This invention relates to an economical process for the bulk production of peptides in the milk of transgenic animals. The production of peptides in milk is ideal as a bulk process because very large volumes of milk can be harvested using simple and environmentally safe technology. A second advantage of using transgenic technology is that only biologically safe materials are produced. This is in contrast to chemical methods where side reactions may produce toxic materials which can only be removed at additional cost.
Another advantage of using a biological process is that some reactions which can be essential for biological activity, for example carboxy-terminal amidation, are difficult to perform in good yield by chemical means. Carboxy-terminal amidation is catalysed by a specific enzyme which recognises and modifies peptides or proteins with a glycine residue at the carboxy terminus ("Peptidylglycine .alpha.-Amidating Monooxygenase: A Multifunctional Protein with Catalytic, Processing and Routing Domains" Eipper, B. A et al. (1993) Protein Science 2, 489-497). Therefore, suitably designed proteins will be specifically amidated before secretion into the milk of producer animals. This is only one example of a range of post-translational modifications which can be carried out by the biosynthetic pathways in the mammary gland and which can potentially be harnessed for the synthesis of particular peptide entities. Other examples of desirable post-translational modifications include disulphide bridge formation, .gamma.-carboxylation of glutamic acid residues and the addition of O- and N-linked glycosylation ("In Vivo Chemical Modification of Proteins", Wold, F., Ann. Rev. Biochem. 50 783-814 (1981)).
The technology for producing large quantities of recombinant proteins, as opposed to shorter peptides, in milk is well established. The human protease inhibitor .alpha..sub.1 -antitrypsin, for example, has been produced in the milk of transgenic sheep at levels in excess of thirty grams of protein per liter ("High Level Expression of Active Human .alpha..sub.1 -Antitrypsin in the Milk of Transgenic Sheep" Wright, G. et al. (1991) Bio/Technology, 9 77-84). It is expected that the same technology can be applied to the production of proteins in cattle which can routinely produce up to 10,000 liters of milk per lactation.